CN112526120A - Method for detecting salbutamol based on SPR technology - Google Patents
Method for detecting salbutamol based on SPR technology Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 30
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 229960002052 salbutamol Drugs 0.000 title claims abstract description 14
- 238000005516 engineering process Methods 0.000 title claims abstract description 8
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 229910052961 molybdenite Inorganic materials 0.000 claims abstract description 11
- 229910052982 molybdenum disulfide Inorganic materials 0.000 claims abstract description 11
- 239000000243 solution Substances 0.000 claims description 41
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 30
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- 235000015277 pork Nutrition 0.000 claims description 8
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- ACTRVOBWPAIOHC-XIXRPRMCSA-N succimer Chemical compound OC(=O)[C@@H](S)[C@@H](S)C(O)=O ACTRVOBWPAIOHC-XIXRPRMCSA-N 0.000 claims 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 abstract description 10
- ACTRVOBWPAIOHC-UHFFFAOYSA-N succimer Chemical compound OC(=O)C(S)C(S)C(O)=O ACTRVOBWPAIOHC-UHFFFAOYSA-N 0.000 abstract description 10
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- BUXRLJCGHZZYNE-UHFFFAOYSA-N 2-amino-5-[1-hydroxy-2-(propan-2-ylamino)ethyl]benzonitrile Chemical compound CC(C)NCC(O)C1=CC=C(N)C(C#N)=C1 BUXRLJCGHZZYNE-UHFFFAOYSA-N 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Malonic acid Chemical compound OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
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- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 1
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 1
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- 229950004407 mabuterol Drugs 0.000 description 1
- JSJCTEKTBOKRST-UHFFFAOYSA-N mabuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(C(F)(F)F)=C1 JSJCTEKTBOKRST-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
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- 239000000203 mixture Substances 0.000 description 1
- CWQXQMHSOZUFJS-UHFFFAOYSA-N molybdenum disulfide Chemical compound S=[Mo]=S CWQXQMHSOZUFJS-UHFFFAOYSA-N 0.000 description 1
- 150000002762 monocarboxylic acid derivatives Chemical class 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 1
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- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000001946 ultra-performance liquid chromatography-mass spectrometry Methods 0.000 description 1
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
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- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/59—Transmissivity
- G01N2021/5903—Transmissivity using surface plasmon resonance [SPR], e.g. extraordinary optical transmission [EOT]
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Abstract
The invention discloses a method for detecting salbutamol based on SPR technology, which adjusts the concentration of surface carboxyl functional groups by introducing thiol SAMs of different sulfydryl and carboxyl, replaces the traditional connecting molecules with dimercaptosuccinic acid (DMSA) of dimercaptodicarboxylic acid, and uses MoS2And AuNPs to enhance SPR signals and improve detection sensitivity of SPR biosensors.
Description
Technical Field
The invention relates to a method for detecting salbutamol based on SPR technology, belonging to the field of chemical analysis.
Background
The surface plasmon resonance technology is primarily applied to the interaction between macromolecules through optical refractive index change, such as the investigation of the affinity between antigen-antibody, receptor-ligand, aptamer-recognition object, but is gradually applied to the field of small molecule detection due to the advantages of rapidness, no mark and high sensitivity. The SPR immunosensor is most widely applied, and the detection mode of the SPR immunosensor is mainly competitive reaction, namely, an antigen or an antibody is fixed on the surface of an SPR chip through a monomolecular layer, then the antigen/antibody and a recognition site on a competitive antibody of a detection target object are introduced, the surface refractive index of the SPR chip is changed along with the difference of the concentration of the target object, and a certain metering relation is formed between the concentration of the target object and the refractive index, so that the target object is quantified.
In SPR immunosensor development, the immobilization of antigen/antibody on SPR chips is a very critical step. The thiol molecule is a bifunctional molecule containing a sulfhydryl group and a carboxyl group, and forms an Au-S bond with gold through the sulfhydryl group to form a monomolecular layer (SAMs) on the surface of a gold film, thereby effectively reducing the nonspecific adsorption of protein on a gold sheet. Meanwhile, carboxyl is covalently coupled with amino in the antigen/antibody by a carbodiimide method, so that the antigen/antibody is bonded on the surface of the chip with the substrate gold film. As a linking molecule, the density of carboxyl groups in thiol greatly affects antigen/antibody binding, and further affects sensor sensitivity and detection performance.
Currently, MPA of monothio monocarboxylic acid is commonly used as the detection SPR chip linking molecule, and in the process of using MPA as SAMs, the density of carboxyl groups is extremely high and far exceeds the active sites required by immobilized antigens, which inevitably affects the antigen/antibody combination, and further affects the sensitivity and detection performance of the sensor.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a method for detecting salbutamol based on SPR technology, wherein the concentration of surface carboxyl functional groups is adjusted by introducing thiol SAMs with different sulfydryl and carboxyl, dimercaptosuccinic acid (DMSA) of dimercaptodicarboxylic acid is used for replacing the traditional connecting molecules, and Mo is used for detecting salbutamolS2And AuNPs are used for enhancing SPR signals, improving the detection sensitivity of the SPR biosensor and providing a new reference for detecting salbutamol.
A method for detecting salbutamol based on SPR technology comprises the following steps:
s1 MoS2-AuNPs-DHLA SPR chip;
s2 adding 10-12mL acetonitrile into 1-1.2g pork, homogenizing, taking supernatant, drying with nitrogen, dissolving with 2-2.3mL n-hexane, adding 1-1.2mL PBS solution, mixing, incubating in 80-85 deg.C water bath for 2-3min, collecting bottom solution, and filtering with filter membrane to obtain liquid to be measured;
s3, preparing a standard curve by using the SAL-Ab solution and SAL standard solutions with different concentrations;
s4 taking 240-250 mu L of liquid to be tested in the step S2 for equipping the MoS manufactured in the step S12SPR immunosensor detection of AuNPs-DHLA SPR chip, and obtaining salbutamol content in pork according to the standard curve prepared in step S3.
Further, the concentration of the SAL-Ab solution in the step S3 is 75 μ g/mL; the concentrations of the SAL standard solutions with different concentrations are 5, 10, 20, 30, 40, 60, 80, 100 and 150ng/mL respectively.
Further, the method for manufacturing the MoS in the step S12-a method of AuNPs-DHLA SPR chip comprising the steps of:
1) taking 10-15mg MoS2Dissolving in 1mL of ionic liquid (1 butyl-3-methylimidazolium hexafluorophosphate), centrifuging once to obtain supernatant A, centrifuging twice to obtain precipitate, washing the precipitate with N, N-Dimethylformamide (DMF) for 3 times, drying, dispersing in dimethylformamide to obtain dispersion with final concentration of 1.8-2 mg/mL-1；
2) Coating 200mL of the dispersion solution 190-200mL prepared in the step 1) on an SPR chip;
3) soaking the chip treated in the step 2) in a 1, 6-Hexanedithiol (HDT) solution, respectively washing the soaked chip once with ethanol and water, drying the chip with nitrogen, putting the treated chip in an AuNPs solution overnight, washing the chip with deionized water, and drying the chip with nitrogen;
4) respectively placing the chips treated in the step 3) in 0.5mmol/L DMSA solution, reacting for 23-24h, washing the SPR chips with ethanol and deionized water respectively after the reaction is finished, and drying with nitrogen;
5) placing the SPR chip treated in the step 4) in a mixed solution of 400 mmol/L1-ethyl- (3-dimethylaminopropyl) carbonyl diimide hydrochloride (EDC) and 100mmol/L N-hydroxysuccinimide (NHS), reacting for 25-30min, and flushing nitrogen with deionized water to blow dry after the reaction is finished;
6) coating 180. mu.L of SAL antigen solution in the SPR chip treated in the step 5), blocking the unblocked unreacted active sites by using ethanolamine solution, finally washing by using PBS solution, and MoS2AuNPs-DHLA SPR chips.
Further, the conditions of the primary centrifugation in the step 1) are as follows: rotating speed 2000rpm for 20 min; the conditions of the secondary centrifugation are as follows: the rotation speed is 6000rpm, and the time is 20 min.
Further, the concentration of the 1, 6-Hexanedithiol (HDT) solution in the step 3) is 0.01mol/L, and the soaking time is 24 h.
Further, the AuNPs solution in step 3) is 25nm AuNPs solution.
Further, the concentration of the DMSA solution in the step 4) is 0.5 mmol/L.
Further, the mixing ratio of 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in the step 5) is 4: 1.
Further, the concentration of the SAL antigen solution in the step 6) is 1 mg/mL.
Has the advantages that:
in the research, the concentration of carboxyl functional groups on the surface is adjusted by introducing thiol SAMs of different sulfydryl and carboxyl, and the traditional connecting molecules such as MPA of mono-sulfydryl monocarboxylic acid are replaced by bis-sulfydryl succinic acid (DMSA) of bis-sulfydryl dicarboxylic acid, so that the problem of steric hindrance in antigen/antibody combination is solved, the configuration and orientation of the antibody are controlled, and the sensitivity is improved.
Drawings
FIG. 1 chip refractive index changes at different SAL-Ab concentrations.
FIG. 2 Standard Curve for detection of SAL by SPR immunosensor.
FIG. 3 DMSA-SPR immunosensor-specific assay.
Detailed Description
In order to make the technical solutions in the present application better understood, the present invention is further described below with reference to examples, which are only a part of examples of the present application, but not all examples, and the present invention is not limited by the following examples.
EXAMPLE 1 preparation of SPR chip
First, experiment method
1、MoS2Modification of SPR chip: 10mg of MoS2 was sonicated at room temperature (22. + -. 2 ℃ C.) for 4h and centrifuged at 2000rpm for 20min to remove large particles. The supernatant was centrifuged at 6000rpm for 20min and then washed 3 times with 1mL of N, N-Dimethylformamide (DMF) each time, and the collected precipitate was dried at 50 deg.C, dispersed in dimethylformamide at a concentration of 2 mg. multidot.mL-1, and stored at 4 deg.C. Draw 200mL of dispersion, coat onto clean SPR dies and dry in air.
2. Modification of SPR chip by AuNPs: firstly, MoS2Soaking the modified SPR chip in 0.01 mol/L1, 6-Hexanedithiol (HDT) solution for 24h, washing the surface of the gold film with ethanol and water, and drying with nitrogen; then the SPR chip is put into a 25nm AuNPs solution overnight, washed clean by deionized water and dried by nitrogen.
3. Modification of the SPR chip by the self-assembled monolayer: the AuNPs modified SPR chip is respectively placed in 0.5mM mercaptopropionic acid (MPA), dihydrolipoic acid (DHLA) and DMSA solution for reaction for 24 h. After the reaction was completed, the SPR chip was rinsed with ethanol and deionized water and dried with nitrogen.
4. Assembly of salbutamol antigen on SPR chip: the SPR chip was placed in a solution of 400mM 1-ethyl- (3-dimethylaminopropyl) carbonyldiimide hydrochloride (EDC) and 100mM N-hydroxysuccinimide (NHS) and reacted at room temperature for 30 min. After the reaction is finished, deionized water is usedThe SPR chip was rinsed with water and dried with nitrogen. The SPR chip treated in (2) was coated with 200. mu.L of SAL antigen (SAL-BSA) solution (1mg/mL), and unreacted active sites were blocked with ethanolamine solution (1.0M, pH 8.5), and finally washed with PBS solution to obtain MoS2-AuNPs-MPA SPR chip, MoS2-AuNPs-DMSA SPR chip and MoS2AuNPs-DHLA SPR chips, sealed at 4 ℃.
5. Selection of optimal SAL monoclonal antibody (SAL-Ab) concentration: different concentrations of SAL-Ab were optimized with 25ug/mL as the optimal reaction concentration.
6. Selection of optimal self-assembled monolayers: study of MoS Using SPR Sensors2-AuNPs-MPA、MoS2-AuNPs-DMSA and MoS2Binding efficiency of antigen/antibody binding on AuNPs-DHLA SPR chips. The change of the response value of the SPR signal generated by flowing SAL-Ab with the same concentration through the surface of the SPR chip is used as a judgment standard.
Second, experimental results
As can be seen from FIG. 1, when the SAL-Ab concentration is 75. mu.g/mL, the concentration of SAL-Ab that can be bound by the three chips is saturated, and at this time, MoS is present2AuNPs-MPA chip (a) and MoS2The refractive index difference between the AuNPs-DMSA chips (b) was 58.8 and 115.6, which indicates that the DMSA chips can bind more SAL-Ab under the same experimental conditions.
Example 2 verification method for detecting salbutamol in sample
1. Sample treatment: adding 10mL of acetonitrile into 1g of pork, homogenizing and extracting for 1min, taking 1mL of supernatant into a 5mL centrifuge tube, drying by using nitrogen, dissolving by using 2mL of n-hexane, then adding 1mL of PBS solution, fully and uniformly oscillating, incubating in a water bath at 85 ℃ for 3min, collecting bottom liquid as test liquid, filtering the liquid to be tested through a 0.22 mu m filter membrane, taking 250 mu L of the liquid to be tested for an SPR immunosensor, and horizontally repeating for 3 times for each sample.
2. And (3) verification of methodology: optimal concentrations of SAL-Ab and different concentrations of SAL standard solution (5, 10, 20, 30, 40, 60, 80, 100, 150ng/mL) were mixed in equal volumes and incubated at 37 ℃ for 30min, each concentration was assayed in 3 replicates, and changes in refractive index were recorded to generate a standard curve.The results show that the method has good linear range and detection sensitivity between 5ng/mL and 150ng/mL, as shown in FIG. 2. Linear regression equation y is 0.008x2The linear correlation coefficient of-1.95 x +114.29 was 0.988, and the detection sensitivity was 5 ng/mL.
To verify the accuracy and reliability of the SPR method, pork was used as a sample substrate, and the addition recovery experiments were performed at concentrations of 5, 10 and 20 μ g/kg (n-3). The results are shown in table 1, the recovery rate of SAL detected by the method is 94.9-108.0%, and RSD is 1.30-5.58%, which has good consistency compared with the results of UPLC-MS/MS method. The method can be used for rapidly detecting salbutamol in actual pork samples.
TABLE 1 DMSA-SPR immunosensor and UPLC/MS/MS assay results for salbutamol in pork samples
In order to examine the recognition specificity of the method, ractopamine, clenbuterol hydrochloride, mabuterol hydrochloride and cimaterol are selected as SAL structural analogues. As shown in FIG. 3, at a concentration of 120ng/mL, the inhibition of SAL was significantly higher than that of the other compounds, and was comparable to that of the mixture. Therefore, the method has good specific recognition performance.
Claims (9)
1. A method for detecting salbutamol based on SPR technology is characterized by comprising the following steps:
s1 MoS2-AuNPs-DHLA SPR chip;
s2 adding 10-12mL acetonitrile into 1-1.2g pork, homogenizing, taking supernatant, drying with nitrogen, dissolving with 2-2.3mL n-hexane, adding 1-1.2mL PBS solution, mixing, incubating in 80-85 deg.C water bath for 2-3min, collecting bottom solution, and filtering with filter membrane to obtain liquid to be measured;
s3, preparing a standard curve by using the SAL-Ab solution and SAL standard solutions with different concentrations;
s4 taking 240-250 mu L of liquid to be tested in the step S2 for being equipped withMoS created in step S12SPR immunosensor detection of AuNPs-DHLA SPR chip, and obtaining salbutamol content in pork according to the standard curve prepared in step S3.
2. The method of claim 1, wherein the SAL-Ab solution of step S3 is at a concentration of 75 μ g/mL; the concentrations of the SAL standard solutions with different concentrations are 5, 10, 20, 30, 40, 60, 80, 100 and 150ng/mL respectively.
3. The method of claim 1, wherein the step S1 of making MoS2-a method of AuNPs-DHLA SPR chip comprising the steps of:
1) taking 10-15mg MoS2Dissolving in 1mL of 1 butyl-3-methylimidazolium hexafluorophosphate ionic liquid, centrifuging once to retain supernatant A, centrifuging twice to retain precipitate, washing the precipitate with N, N-dimethylformamide for 3 times, drying, dispersing in dimethylformamide to obtain dispersion with final concentration of 1.8-2 mg/mL-1；
2) Coating 200mL of the dispersion solution 190-200mL prepared in the step 1) on an SPR chip;
3) soaking the chip treated in the step 2) in a 1, 6-hexanedithiol solution, respectively washing the soaked chip once by using ethanol and water, drying the chip by using nitrogen, putting the treated chip in an AuNPs solution overnight, washing the chip by using deionized water, and drying the chip by using nitrogen;
4) respectively placing the chips treated in the step 3) in 0.5mmol/L DMSA solution, reacting for 23-24h, washing the SPR chips with ethanol and deionized water respectively after the reaction is finished, and drying with nitrogen;
5) placing the SPR chip treated in the step 4) in a mixed solution of 400 mmol/L1-ethyl- (3-dimethylaminopropyl) carbonyl diimide hydrochloride and 100mmol/L N-hydroxysuccinimide, reacting for 25-30min, and flushing with deionized water and drying with nitrogen after the reaction is finished;
6) coating 180 and 200 mu L of SAL antigen solution in the SPR chip treated in the step 5), blocking the unblocked unreacted active sites by using ethanolamine solution,finally rinsing with PBS solution, MoS2AuNPs-DHLA SPR chips.
4. The method of claim 3, wherein the conditions of one centrifugation in step 1) are: rotating speed 2000rpm for 20 min; the conditions of the secondary centrifugation are as follows: the rotation speed is 6000rpm, and the time is 20 min.
5. The method according to claim 3, wherein the concentration of the 1, 6-hexanedithiol solution in step 3) is 0.01mol/L and the soaking time is 24 h.
6. The method of claim 3, wherein the AuNPs solution in step 3) is a 25nm AuNPs solution.
7. The method of claim 3, wherein the concentration of the DMSA solution in step 4) is 0.5 mmol/L.
8. The method of claim 3, wherein the 1-ethyl- (3-dimethylaminopropyl) carbonyldiimidate hydrochloride and N-hydroxysuccinimide are mixed in a ratio of 4:1 in step 5).
9. The method of any one of claims 3-8, wherein the concentration of the SAL antigen solution in step 6) is 1 mg/mL.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202011298840.2A CN112526120B (en) | 2020-11-19 | 2020-11-19 | Method for detecting salbutamol based on SPR technology |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115615963A (en) * | 2022-08-15 | 2023-01-17 | 中国农业科学院农业质量标准与检测技术研究所 | Method for rapidly determining salbutamol residue in animal urine without marking SPR technology |
Citations (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2737500A (en) * | 1950-11-30 | 1956-03-06 | Monsanto Chemicals | Process of precipitating anionic substances and precipitated compositions |
| US20070065954A1 (en) * | 2005-09-15 | 2007-03-22 | Minoru Taya | Surface plasmon resonance biosensor system for detection of antigens and method for determining the presence of antigens |
| CN102191034A (en) * | 2010-03-03 | 2011-09-21 | 中国科学技术大学 | N-(4-aminobutyl)-N-ethylisoluminol luminescence functionalized nanogold, and preparation method and application thereof |
| CN102556959A (en) * | 2011-12-30 | 2012-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Preparation method of metal nanoparticle dimer |
| CN102559170A (en) * | 2010-12-30 | 2012-07-11 | 中国科学技术大学 | Luminol-luminescent functionalized nano-silver as well as preparation method and application for same |
| CN103011292A (en) * | 2012-12-04 | 2013-04-03 | 北京大学 | Nolybdenum disulfide nanometer particle, preparation method and application thereof |
| JP2015118911A (en) * | 2013-12-19 | 2015-06-25 | 深▲セン▼市貝特瑞新能源材料股▲ふん▼有限公司 | Silicon-based composite negative electrode material for lithium ion secondary batteries, manufacturing method, and battery |
| US20160333383A1 (en) * | 2015-05-14 | 2016-11-17 | Institute of Agro-Products Processing Science and Technology, CAAS | Method for Preparing Yeast Beta-D-Glucan Using Solubilization Technology Based on Molecular Assembly |
| CN106917131A (en) * | 2017-02-28 | 2017-07-04 | 湖北大学 | A kind of preparation method of shitosan/molybdenum bisuphide photocatalysis antibacterial coating |
| CN107244697A (en) * | 2017-07-29 | 2017-10-13 | 深圳孔雀科技开发有限公司 | A kind of preparation method of individual layer molybdenum disulfide nano sheet |
| CN107300548A (en) * | 2017-06-19 | 2017-10-27 | 华中科技大学 | A kind of flexible surface enhancing Raman substrate material and preparation method and application |
| CN107849118A (en) * | 2015-02-19 | 2018-03-27 | 艾奥尼卡科学 | For detecting the reagent and method of infectious diseases |
| CN108760852A (en) * | 2018-04-13 | 2018-11-06 | 江西师范大学 | Photoelectrochemical ochratoxin A detection method based on dual signal amplification |
| CN108918856A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors |
| CN109142476A (en) * | 2018-08-14 | 2019-01-04 | 青岛科技大学 | Complex film modified electrode of functionalization molybdenum disulfide nano sheet and preparation method thereof and detection application |
| CN109256546A (en) * | 2018-09-03 | 2019-01-22 | 山西煤炭进出口集团科学技术研究院有限公司 | A kind of molybdenum disulfide/graphene composite material and its preparation method and application |
| US20200025752A1 (en) * | 2017-09-01 | 2020-01-23 | California Institute Of Technology | Method for organizing individual molecules on a patterned substrate and structures assembled thereby |
| CN110772648A (en) * | 2019-10-11 | 2020-02-11 | 东华大学 | Preparation method of RGD modified dendrimer coated CuS nanoparticles |
| CN111715243A (en) * | 2020-06-18 | 2020-09-29 | 扬州大学 | Autocatalysis-heterojunction photoelectric composite material and preparation and sensing application thereof |
| CA3136161A1 (en) * | 2019-04-16 | 2020-10-22 | Laboratory Corporation Of America Holdings | Methods and systems for detecting antibodies by surface plasmon resonance |
-
2020
- 2020-11-19 CN CN202011298840.2A patent/CN112526120B/en active Active
Patent Citations (21)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US2737500A (en) * | 1950-11-30 | 1956-03-06 | Monsanto Chemicals | Process of precipitating anionic substances and precipitated compositions |
| US20070065954A1 (en) * | 2005-09-15 | 2007-03-22 | Minoru Taya | Surface plasmon resonance biosensor system for detection of antigens and method for determining the presence of antigens |
| CN102191034A (en) * | 2010-03-03 | 2011-09-21 | 中国科学技术大学 | N-(4-aminobutyl)-N-ethylisoluminol luminescence functionalized nanogold, and preparation method and application thereof |
| CN102362182A (en) * | 2010-03-03 | 2012-02-22 | 中国科学技术大学 | N-(4-aminobutyl)-n-ethylisoluminol functionalized gold nanoparticles, preparation method and application thereof |
| CN102559170A (en) * | 2010-12-30 | 2012-07-11 | 中国科学技术大学 | Luminol-luminescent functionalized nano-silver as well as preparation method and application for same |
| CN102556959A (en) * | 2011-12-30 | 2012-07-11 | 中国科学院苏州纳米技术与纳米仿生研究所 | Preparation method of metal nanoparticle dimer |
| CN103011292A (en) * | 2012-12-04 | 2013-04-03 | 北京大学 | Nolybdenum disulfide nanometer particle, preparation method and application thereof |
| JP2015118911A (en) * | 2013-12-19 | 2015-06-25 | 深▲セン▼市貝特瑞新能源材料股▲ふん▼有限公司 | Silicon-based composite negative electrode material for lithium ion secondary batteries, manufacturing method, and battery |
| CN107849118A (en) * | 2015-02-19 | 2018-03-27 | 艾奥尼卡科学 | For detecting the reagent and method of infectious diseases |
| US20160333383A1 (en) * | 2015-05-14 | 2016-11-17 | Institute of Agro-Products Processing Science and Technology, CAAS | Method for Preparing Yeast Beta-D-Glucan Using Solubilization Technology Based on Molecular Assembly |
| CN106917131A (en) * | 2017-02-28 | 2017-07-04 | 湖北大学 | A kind of preparation method of shitosan/molybdenum bisuphide photocatalysis antibacterial coating |
| CN107300548A (en) * | 2017-06-19 | 2017-10-27 | 华中科技大学 | A kind of flexible surface enhancing Raman substrate material and preparation method and application |
| CN107244697A (en) * | 2017-07-29 | 2017-10-13 | 深圳孔雀科技开发有限公司 | A kind of preparation method of individual layer molybdenum disulfide nano sheet |
| US20200025752A1 (en) * | 2017-09-01 | 2020-01-23 | California Institute Of Technology | Method for organizing individual molecules on a patterned substrate and structures assembled thereby |
| CN108760852A (en) * | 2018-04-13 | 2018-11-06 | 江西师范大学 | Photoelectrochemical ochratoxin A detection method based on dual signal amplification |
| CN108918856A (en) * | 2018-07-31 | 2018-11-30 | 济南大学 | A kind of preparation method and application of double MOFs material quenching type electrochemiluminescimmunosensor immunosensors |
| CN109142476A (en) * | 2018-08-14 | 2019-01-04 | 青岛科技大学 | Complex film modified electrode of functionalization molybdenum disulfide nano sheet and preparation method thereof and detection application |
| CN109256546A (en) * | 2018-09-03 | 2019-01-22 | 山西煤炭进出口集团科学技术研究院有限公司 | A kind of molybdenum disulfide/graphene composite material and its preparation method and application |
| CA3136161A1 (en) * | 2019-04-16 | 2020-10-22 | Laboratory Corporation Of America Holdings | Methods and systems for detecting antibodies by surface plasmon resonance |
| CN110772648A (en) * | 2019-10-11 | 2020-02-11 | 东华大学 | Preparation method of RGD modified dendrimer coated CuS nanoparticles |
| CN111715243A (en) * | 2020-06-18 | 2020-09-29 | 扬州大学 | Autocatalysis-heterojunction photoelectric composite material and preparation and sensing application thereof |
Non-Patent Citations (5)
| Title |
|---|
| ZONGYOU YIN 等: "Au Nanoparticle-Modified MoS2 Nanosheet-Based Photoelectrochemical Cells for Water Splitting", SMALL, vol. 10, no. 17 * |
| 张琦 等: "单层二硫化钼纳米片负载金纳米颗粒用于等离子共振耦合增强的近红外光热治疗", 中国化学会第30届学术年会摘要集-第三十八分会：纳米生物效应与纳米药物化学, pages 1 * |
| 李文杰 等: "基于DMSA自组装单分子层的SPR免疫传感器检测沙丁胺醇", 食品工业科技, vol. 41, no. 22, pages 2 - 3 * |
| 王晨晨 等: "表面等离子体共振技术检测沙丁胺醇", 分析试验室, vol. 38, no. 8, pages 1 - 2 * |
| 谭婷婷;王光寅;潘祖亭;罗运柏;: "纳米材料在生物检测中的应用", 化学与生物工程, no. 03, 25 March 2009 (2009-03-25) * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115615963A (en) * | 2022-08-15 | 2023-01-17 | 中国农业科学院农业质量标准与检测技术研究所 | Method for rapidly determining salbutamol residue in animal urine without marking SPR technology |
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